Aggregation Of Huntingtin Protein Research Articles

Two novel DnaJ chaperone proteins CG5001 and P58IPK regulate the pathogenicity of Huntington’s disease related aggregates

Huntington’s disease (HD) is a rare neurodegenerative disease caused due to aggregation of Huntingtin (HTT) protein. This study involves the cloning of 40 DnaJ chaperones from Drosophila, and overexpressing them in yeasts and fly models of HD. Accordingly, DnaJ chaperones were catalogued as enhancers or suppressors based on their growth phenotypes and aggregation properties. 2 of the chaperones that came up as targets were CG5001 and P58IPK. Protein aggregation and slow growth phenotype was rescued in yeasts, S2 cells, and Drosophila transgenic lines of HTT103Q with these overexpressed chaperones. Since DnaJ chaperones have protein sequence similarity across species, they can be used as possible tools to combat the effects of neurodegenerative diseases.

Glymphatic influx and clearance are perturbed in Huntington's disease.

The accumulation of mutant huntingtin protein aggregates in neurons is a pathological hallmark of Huntington's disease (HD). The glymphatic system, a brain-wide perivascular network, facilitates the exchange of interstitial fluid (ISF) and cerebrospinal fluid (CSF), supporting interstitial solute clearance of brain wastes. In this study, we employed dynamic glucose-enhanced (DGE) MRI to measure D-glucose clearance from CSF as a tool to predict glymphatic function in a mouse model of HD. We found significantly diminished CSF clearance efficiency in HD mice prior to phenotypic onset. The impairment of CSF clearance efficiency worsened with disease progression. These DGE MRI findings in compromised glymphatic function were further confirmed with fluorescence-based imaging of CSF tracer influx, suggesting an impaired glymphatic function in premanifest HD. Moreover, expression of the astroglial water channel aquaporin-4 (AQP4) in the perivascular compartment, a key mediator of glymphatic function, was significantly diminished in both HD mouse brain and human HD brain. Our data, acquired using a clinically translatable MRI, indicate a perturbed glymphatic network in the HD brain. Further validation of these findings in clinical studies will provide insights into the potential of glymphatic clearance as a therapeutic target as well as an early biomarker in HD.

In vivo CRISPR base editing for treatment of Huntington's disease.

Huntington's disease (HD) is an inherited and ultimately fatal neurodegenerative disorder caused by an expanded polyglutamine-encoding CAG repeat within exon 1 of the huntingtin (HTT) gene, which produces a mutant protein that destroys striatal and cortical neurons. Importantly, a critical event in the pathogenesis of HD is the proteolytic cleavage of the mutant HTT protein by caspase-6, which generates fragments of the N-terminal domain of the protein that form highly toxic aggregates. Given the role that proteolysis of the mutant HTT protein plays in HD, strategies for preventing this process hold potential for treating the disorder. By screening 141 CRISPR base editor variants targeting splice elements in the HTT gene, we identified platforms capable of producing HTT protein isoforms resistant to caspase-6-mediated proteolysis via editing of the splice acceptor sequence for exon 13. When delivered to the striatum of a rodent HD model, these base editors induced efficient exon skipping and decreased the formation of the N-terminal fragments, which in turn reduced HTT protein aggregation and attenuated striatal and cortical atrophy. Collectively, these results illustrate the potential for CRISPR base editing to decrease the toxicity of the mutant HTT protein for HD.

Polyphenol-based modulation of structure and toxicity of huntingtin protein aggregates

Polyphenol-based modulation of structure and toxicity of huntingtin protein aggregates

Autophagy-Induced Nanoparticle-Based Clearing of Toxic Huntingtin Protein Aggregates from Neuron Cells

Autophagy-Induced Nanoparticle-Based Clearing of Toxic Huntingtin Protein Aggregates from Neuron Cells

A method for the analysis of the oligomerization profile of the Huntington's disease-associated, aggregation-prone mutant huntingtin protein by isopycnic ultracentrifugation.

Conformational diseases, such as Alzheimer's, Parkinson's and Huntington's diseases as well as ataxias and fronto-temporal disorders, are part of common class of neurological disorders characterised by the aggregation and progressive accumulation of mutant proteins which display aberrant conformation. In particular, Huntington's disease (HD) is caused by mutations leading to an abnormal expansion in the polyglutamine (poly-Q) tract of the huntingtin protein (HTT), leading to the formation of inclusion bodies in neurons of affected patients. Furthermore, recent experimental evidence is challenging the conventional view of the disease by revealing the ability of mutant HTT to be transferred between cells by means of extracellular vesicles (EVs), allowing the mutant protein to seed oligomers involving both the mutant and wild type forms of the protein. There is still no successful strategy to treat HD. In addition, the current understanding of the biological processes leading to the oligomerization and aggregation of proteins bearing the poly-Q tract has been derived from studies conducted on isolated poly-Q monomers and oligomers, whose structural properties are still unclear and often inconsistent. Here we describe a standardised biochemical approach to analyse by isopycnic ultracentrifugation the oligomerization of the N-terminal fragment of mutant HTT. The dynamic range of our method allows one to detect large and heterogeneous HTT complexes. Hence, it could be harnessed for the identification of novel molecular determinants responsible for the aggregation and the prion-like spreading properties of HTT in the context of HD. Equally, it provides a tool to test novel small molecules or bioactive compounds designed to inhibit the aggregation of mutant HTT.

Imaging and Assay of the Dynamics of Cytotoxic Huntingtin (HTT) Protein Aggregates Regulated by lncRNAs.

Huntington's disease (HD) pathogenesis involves deregulation of coding and noncoding RNA transcripts of which the involvement of long noncoding RNAs (lncRNA) has been realized recently. Of these, Meg3, Neat1, and Xist showed a consistent and significant increase in HD cell and animal models. In the present study, we formulate a methodology to visualize and quantify intracellular aggregates formed by mutant HTT protein. This method employs the use of both confocal laser scanning and super resolution (N-SIM) microscopy to accurately estimate aggregate numbers. Further, to determine the role of two lncRNAs Meg3 and Neat1 in the formation of aggregates of mutant HTT, we used commercially available siRNAs against Meg3 and Neat1 for transiently knocking them down in mouse Neuro2a and human SHSY5Y cells. Co-transfection of 83Q-DsRed and siRNA specific for Neat1 or Meg3 resulted in decreased intracellular aggregates of 83Q-DsRed in both the cell lines. We have established a quantitative method to estimate and directly or indirectly modulate the formation of mutant HTTaggregates.

Imaging of intracellular protein aggregates through plasmon-assisted clusteroluminescence.

The formation of clusters in non-aromatic molecules can give rise to unconventional luminescence or clusteroluminescence. Typically containing heteroatoms without extended conjugation or aromatic rings, these molecules have drawn much attention owing to the prospects of label-free biological imaging. However, their applications have been limited due to the lack of knowledge of the underlying mechanism. Herein, we have elucidated the mechanism of clusteroluminescence from proteins, which were explicitly aggregated using plasmonic silver nanoparticles. The nanoparticles promoted protein aggregation and induced nitrile formation on the surface, which, along with other lone-pair-containing heteroatoms, contributed to enhanced emission in the visible range. Remarkably, this makes imaging of proteins possible with visible excitations, as co-factor-lacking proteins generally undergo electronic transitions only in the ultraviolet range. Furthermore, the inherent protein-aggregating behaviour of plasmonic nanoparticles was harnessed for imaging of intracellular Huntingtin protein aggregates overexpressed in HeLa cells through clusteroluminescence. Significant plasmon-enhanced and red-shifted fluorescence emission was observed, which helped in the imaging and localization of the intracellular aggregates. Density functional theory calculations and transient absorbance spectroscopy were used to probe the molecular interactions at the protein-nanoparticle interface and the charge transfer states, further elucidating the role of nanoparticles and the emission mechanism. This technique thus opens alternate avenues for label-free fluorescence bioimaging.

Interaction between huntingtin exon 1 and HEAT repeat structure probed by chimeric model proteins.

Huntington disease (HD) is associated with aggregation of huntingtin (HTT) protein containing over 35 continuous Q residues within the N-terminal exon 1 encoded region. The C-terminal of the HTT protein consists mainly of HEAT repeat structure which serves as a scaffold for multiple cellular activities. Structural and biochemical analysis of the intact HTT protein has been hampered by its huge size (~300 kDa) and most in vitro studies to date have focused on the properties of the exon 1 region. To explore the interaction between HTT exon 1 and the HEAT repeat structure, we constructed chimeric proteins containing the N-terminal HTT exon 1 region and the HEAT repeat protein PR65/A. The results indicate that HTT exon 1 slightly destabilizes the downstream HEAT repeat structure and endows the HEAT repeat structure with more conformational flexibility. Wild-type and pathological lengths of polyQ did not show differences in the interaction between HTT exon 1 and the HEAT repeats. With the C-terminal fusion of PR65/A, HTT exon 1 containing pathological lengths of polyQ could still form amyloid fibrils, but the higher-order architecture of fibrils and kinetics of fibril formation were affected by the C-terminal fusion of HEAT repeats. This indicates that interaction between HTT exon 1 and HEAT repeat structure is compatible with both normal function of HTT protein and the pathogenesis of HD, and this study provides a potential model for further exploration.

Charge within Nt17 peptides modulates huntingtin aggregation and initial lipid binding events

Toxic aggregation of pathogenic huntingtin protein (htt) is implicated in Huntington's disease and influenced by various factors, including the first seventeen amino acids at the N-terminus (Nt17) and the presence of lipid membranes. Nt17 has a propensity to form an amphipathic α-helix in the presence of binding partners, which promotes α-helix rich oligomer formation and facilitates htt/lipid interactions. Within Nt17 are multiple sites that are subject to post-translational modification, including acetylation and phosphorylation. Acetylation can occur at lysine 6, 9, and/or 15 while phosphorylation can occur at threonine 3, serine 13, and/or serine 16. Such modifications impact aggregation and lipid binding through the alteration of various intra- and intermolecular interactions. When incubated with htt-exon1(46Q), free Nt17 peptides containing point mutations mimicking acetylation or phosphorylation reduced fibril formation and altered oligomer morphologies. Upon exposure to lipid vesicles, changes to peptide/lipid complexation were observed and peptide-containing oligomers demonstrated reduced lipid interactions.

Investigating the Effects of Brain Cholesterol Metabolism Using CYP46A1 Gene Therapy in Subjects with Huntington’s Disease (P1-11.016)

<h3>Objective:</h3> To report the clinical trial study design evaluating the safety, tolerability, and preliminary efficacy of investigational gene therapy rAAVrh10.CYP46A1wt (AB-1001) in subjects with Huntington’s disease (HD). <h3>Background:</h3> HD is a progressive, genetic neurodegenerative disorder linked to cholesterol dysregulation and subsequent protein aggregation in the brain. A neuron-specific enzyme, cholesterol-24-hydroxylase (CYP46A1), assists in regulating brain cholesterol synthesis and metabolism, and is deficient in subjects with HD. CYP46A1 restoration in striatal neurons using the adeno-associated virus (AAV) therapy AB-1001 led to reduced mutant Huntingtin protein aggregation and improved pathology hallmarks in two mouse models. <h3>Design/Methods:</h3> This first in-human, Phase 1/2, open-label, dose-finding study will consist of one-time intracerebral bilateral injections of AB-1001, an investigational AAV gene therapy comprising an AAVrh10 vector carrying gene encoded CYP46A1, within the striatum. To be eligible for enrollment, subjects will be 18–65 years old, possess a CAG expansion in the huntingtin gene ≥40, have early manifest HD, and have striatal MRI volumes per hemisphere compatible with local delivery of study drug. The trial will include screening (8 weeks), treatment and initial follow-up (52 weeks), and long-term follow-up (4 years). The treatment phase includes dose-finding (Cohort 1: n=3–6, single dose of 4.0E8 vg/μL; Cohort 2: n=3–6, single dose of 1.1E9 vg/μL) and dose-expansion (n=6, nonsequential dosing with selected dose). The primary endpoint will be the incidence of dose-limiting toxicities, treatment-emergent adverse events, and serious adverse events. Secondary endpoints include change from baseline to week 52 in MRI-assessed measures of HD-impacted brain regions, composite Unified Huntington’s Disease Rating Scale (cUHDRS), and MRS metabolic profile. Other endpoints include additional HD biomarkers and quality-of-life measures. <h3>Results:</h3> N/A <h3>Conclusions:</h3> Preclinical results show the potential for CYP46A1 gene therapy with AB-1001 to demonstrate therapeutic benefits in subjects with HD and rationalize progression to the first in-human Phase I/II clinical trial. <b>Disclosure:</b> Dr. Schultz has received personal compensation for serving as an employee of AskBio. The institution of Dr. Durr has received personal compensation in the range of $500-$4,999 for serving as a Consultant for UCB. The institution of Dr. Durr has received personal compensation in the range of $0-$499 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Neurology Genetics. The institution of Dr. Durr has received research support from NIH. The institution of Dr. Durr has received research support from BIOGEN. The institution of Dr. Durr has received research support from MINORYX. The institution of Dr. Durr has received research support from TRIPLETS THERAPEUTICS. The institution of Dr. Durr has received research support from Servier. The institution of Dr. Durr has received research support from ANR. The institution of Dr. Durr has received research support from PTC. Prof. Karachi has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Boston scientific / Medtronic / Neurotrials. Dr. O’Callaghan has received personal compensation for serving as an employee of Asklepios Biopharmaceuticals. Dr. O’Callaghan has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for Asklepios Biopharmaceuticals. Dr. O’Callaghan has stock in Asklepios Biopharmaceuticals. Dr. O’Callaghan has received intellectual property interests from a discovery or technology relating to health care. Dr. ALVES has nothing to disclose. Dr. Lamontagne has received personal compensation for serving as an employee of Asklepios Biopharmaceutical Inc. Mrs. HURMIC has nothing to disclose. Dr. Cartier has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for askbio. Dr. Cartier has stock in brainvectis.

Understanding the role of the hematopoietic niche in Huntington's disease's phenotypic expression: in vivo evidence using a parabiosis model

In a previous study, we have shown that parabiotic coupling of a knock-in mouse model (zQ175) of Huntington's disease (HD) to wild-type (WT) littermates resulted in a worsening of the normal phenotype as seen by detection of mutant huntingtin protein (mHTT) aggregates within peripheral organs and the cerebral cortex as well as vascular abnormalities in WT mice. In contrast, parabiosis improved disease features in the zQ175 mice such as reduction of mHTT aggregate number in the liver and cortex, decrease in blood-brain barrier (BBB) permeability and attenuation of mitochondrial impairments. While the shared circulation mediated these effects, no specific factor was identified. To better understand which blood elements were involved in the aforementioned changes, WT and zQ175 mice underwent parabiotic surgery prior to exposing one of the paired animals to irradiation. The irradiation procedure successfully eliminated the hematopoietic niche followed by repopulation with cells originating from the non-irradiated parabiont, as measured by the quantification of mHTT levels in peripheral blood mononuclear cells. Although irradiation of the WT parabiont, causing the loss of healthy hematopoietic cells, did lead to a few alterations in mitochondrial function in the muscle (TOM40 levels), and increased neuroinflammation in the striatum (GFAP levels), most of the changes observed were likely attributable to the irradiation procedure itself (e.g. mHTT aggregates in cortex and liver; cellular stress in peripheral organs). However, factors such as mHTT aggregation in the brain and periphery, and BBB leakage, which were improved in zQ175 mice when paired to WT littermates in the previous parabiosis experiment, were unaffected by perturbation of the hematopoietic niche. It would therefore appear that cells of the hematopoietic stem cell niche are largely uninvolved in the beneficial effects of parabiosis.

G3BP1-dependent mechanism suppressing protein aggregation in Huntington's models and its demise upon stress granule assembly.

Stress granules are membrane-less ribonucleoprotein organelles that assemble upon exposure to stress conditions, but rapidly disassemble upon removal of stress. However, chronic stress can lead to persistent stress granules, a feature of distinct age-related neurodegenerative disorders. Among them, Huntington's disease (HD), which is caused by mutant expansion of the polyglutamine (polyQ) repeats of huntingtin protein (HTT), leading to its aggregation. To identify modulators of mutant HTT aggregation, we define its interactome in striatal neurons differentiated from patient-derived induced pluripotent stem cells (HD-iPSCs). We find that HTT interacts with G3BP1, a characteristic component of stress granules. Knockdown of G3BP1 increases mutant HTT protein levels and abolishes the ability of iPSCs as well as their differentiated neural counterparts to suppress mutant HTT aggregation. Moreover, loss of G3BP1 hastens polyQ-expanded aggregation and toxicity in the neurons of HD C. elegans models. Likewise, the assembly of G3BP1 into stress granules upon distinct stress conditions also reduces its interaction with HTT in human cells, promoting mutant HTT aggregation. Notably, enhancing the levels of G3BP1 is sufficient to induce proteasomal degradation of mutant HTT and prevent its aggregation, whereas the formation of stress granules blocks these ameliorative effects. In contrast, a mutant G3BP1 variant that cannot accumulate into granules retains its capacity to prevent mutant HTT aggregation even when the cells assemble stress granules. Thus, our findings indicate a direct role of G3BP1 and stress granule assembly in mutant HTT aggregation that may have implications for HD.

PolyQ-Expanded Mutant Huntingtin Forms Inclusion Body Following Transient Cold Shock in a Two-Step Aggregation Mechanism.

Age-dependent formation of insoluble protein aggregates is a hallmark of many neurodegenerative diseases. We are interested in the cell chemistry that drives the aggregation of polyQ-expanded mutant Huntingtin (mHtt) protein into insoluble inclusion bodies (IBs). Using an inducible cell model of Huntington's disease, we show that a transient cold shock (CS) at 4 °C followed by recovery incubation at temperatures of 25-37 °C strongly and rapidly induces the compaction of diffuse polyQ-expanded HuntingtinExon1-enhanced green fluorescent protein chimera protein (mHtt) into round, micron size, cytosolic IBs. This transient CS-induced mHtt IB formation is independent of microtubule integrity or de novo protein synthesis. The addition of millimolar concentrations of sodium chloride accelerates, whereas urea suppresses this transient CS-induced mHtt IB formation. These results suggest that the low temperature of CS constrains the conformation dynamics of the intrinsically disordered mHtt into labile intermediate structures to facilitate de-solvation and hydrophobic interaction for IB formation at the higher recovery temperature. This work, along with our previous observation of the effects of heat shock protein chaperones and osmolytes in driving mHtt IB formation, underscores the primacy of mHtt structuring and rigidification for H-bond-mediated cross-linking in a two-step mechanism of mHtt IB formation in living cells.

Design and Evaluation of [18F]CHDI-650 as a Positron Emission Tomography Ligand to Image Mutant Huntingtin Aggregates.

Therapeutic interventions are being developed for Huntington's disease (HD), a hallmark of which is mutant huntingtin protein (mHTT) aggregates. Following the advancement to human testing of two [11C]-PET ligands for aggregated mHTT, attributes for further optimization were identified. We replaced the pyridazinone ring of CHDI-180 with a pyrimidine ring and minimized off-target binding using brain homogenate derived from Alzheimer's disease patients. The major in vivo metabolic pathway via aldehyde oxidase was blocked with a 2-methyl group on the pyrimidine ring. A strategically placed ring-nitrogen on the benzoxazole core ensured high free fraction in the brain without introducing efflux. Replacing a methoxy pendant with a fluoro-ethoxy group and introducing deuterium atoms suppressed oxidative defluorination and accumulation of [18F]-signal in bones. The resulting PET ligand, CHDI-650, shows a rapid brain uptake and washout profile in non-human primates and is now being advanced to human testing.

An RNA-targeting CRISPR–Cas13d system alleviates disease-related phenotypes in Huntington’s disease models

Huntington’s disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Since the reduction of pathogenic mutant HTT messenger RNA is therapeutic, we developed a mutant allele-sensitive CAGEX RNA-targeting CRISPR–Cas13d system (Cas13d–CAGEX) that eliminates toxic CAGEX RNA in fibroblasts derived from patients with HD and induced pluripotent stem cell-derived neurons. We show that intrastriatal delivery of Cas13d–CAGEX via an adeno-associated viral vector selectively reduces mutant HTT mRNA and protein levels in the striatum of heterozygous zQ175 mice, a model of HD. This also led to improved motor coordination, attenuated striatal atrophy and reduction of mutant HTT protein aggregates. These phenotypic improvements lasted for at least eight months without adverse effects and with minimal off-target transcriptomic effects. Taken together, we demonstrate proof of principle of an RNA-targeting CRISPR–Cas13d system as a therapeutic approach for HD, a strategy with implications for the treatment of other dominantly inherited disorders.

Regulation of Blos1 by IRE1 prevents the accumulation of Huntingtin protein aggregates.

Huntington's disease is characterized by accumulation of the aggregation-prone mutant Huntingtin (mHTT) protein. Here, we show that expression of exon 1 of mHTT in mouse cultured cells activates IRE1, the transmembrane sensor of stress in the endoplasmic reticulum, leading to degradation of the Blos1 mRNA and repositioning of lysosomes and late endosomes toward the microtubule organizing center. Overriding Blos1 degradation results in excessive accumulation of mHTT aggregates in both cultured cells and primary neurons. Although mHTT is degraded by macroautophagy when highly expressed, we show that before the formation of large aggregates, mHTT is degraded via an ESCRT-dependent, macroautophagy-independent pathway consistent with endosomal microautophagy. This pathway is enhanced by Blos1 degradation and appears to protect cells from a toxic, less aggregated form of mHTT.

Biodistribution and dosimetry in human healthy volunteers of the PET radioligands [11C]CHDI-00485180-R and [11C]CHDI-00485626, designed for quantification of cerebral aggregated mutant huntingtin.

Huntington's disease is caused by a trinucleotide expansion in the HTT gene, which leads to aggregation of mutant huntingtin (mHTT) protein in the brain and neurotoxicity. Direct in vivo measurement of mHTT aggregates in human brain parenchyma is not yet possible. In this first-in-human study, we investigated biodistribution and dosimetry in healthy volunteers of [11C]CHDI-00485180-R ([11C]CHDI-180R) and [11C]CHDI-00485626 ([11C]CHDI-626), two tracers designed for PET imaging of aggregated mHTT in the brain that have been validated in preclinical models. Biodistribution and radiation dosimetry studies were performed in 3 healthy volunteers (age 25.7 ± 0.5years; 2 F) for [11C]CHDI-180R and in 3 healthy volunteers (age 35.3 ± 6.8years; 2 F) for [11C]CHDI-626 using sequential whole-body PET-CT. Source organs were delineated in 3D using combined PET and CT data. Individual organ doses and effective doses were determined using OLINDA 2.1. There were no clinically relevant adverse events. The mean effective dose (ED) for [11C]CHDI-180R was 4.58 ± 0.65 μSv/MBq, with highest absorbed doses for liver (16.9μGy/MBq), heart wall (15.9μGy/MBq) and small intestine (15.8μGy/MBq). Mean ED for [11C]CHDI-626 was 5.09 ± 0.06 μSv/MBq with the highest absorbed doses for the gallbladder (26.5μGy/MBq), small intestine (20.4μGy/MBq) and liver (19.6μGy/MBq). Decay-corrected brain uptake curves showed promising kinetics for [11C]CHDI-180R, but for [11C]CHDI-626 an increasing signal over time was found, probably due to accumulation of a brain-penetrant metabolite. [11C]CHDI-180R and [11C]CHDI-626 are safe for in vivo PET imaging in humans. The estimated radiation burden is in line with most 11C-ligands. While [11C]CHDI-180R has promising kinetic properties in the brain, [11C]CHDI-626 is not suitable for human in vivo mHTT PET due to the possibility of a radiometabolite accumulating in brain parenchyma. EudraCT number 2020-002129-27. gov NCT05224115 (retrospectively registered).

Alternative processing of human HTT mRNA with implications for Huntington's disease therapeutics.

Huntington disease is caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT) that is translated into a polyglutamine stretch in the huntingtin protein (HTT). We previously showed that HTT mRNA carrying an expanded CAG repeat was incompletely spliced to generate HTT1a, an exon 1 only transcript, which was translated to produce the highly aggregation-prone and pathogenic exon 1 HTT protein. This occurred in all knock-in mouse models of Huntington's disease and could be detected in patient cell lines and post-mortem brains. To extend these findings to a model system expressing human HTT, we took advantage of YAC128 mice that are transgenic for a yeast artificial chromosome carrying human HTT with an expanded CAG repeat. We discovered that the HTT1a transcript could be detected throughout the brains of YAC128 mice. We implemented RNAscope to visualize HTT transcripts at the single molecule level and found that full-length HTT and HTT1a were retained together in large nuclear RNA clusters, as well as being present as single transcripts in the cytoplasm. Homogeneous time-resolved fluorescence analysis demonstrated that the HTT1a transcript had been translated to produce the exon 1 HTT protein. The levels of exon 1 HTT in YAC128 mice, correlated with HTT aggregation, supportive of the hypothesis that exon 1 HTT initiates the aggregation process. Huntingtin-lowering strategies are a major focus of therapeutic development for Huntington's disease. These approaches often target full-length HTT alone and would not be expected to reduce pathogenic exon 1 HTT levels. We have established YAC128 mouse embryonic fibroblast lines and shown that, together with our QuantiGene multiplex assay, these provide an effective screening tool for agents that target HTT transcripts. The effects of current targeting strategies on nuclear RNA clusters are unknown, structures that may have a pathogenic role or alternatively could be protective by retaining HTT1a in the nucleus and preventing it from being translated. In light of recently halted antisense oligonucleotide trials, it is vital that agents targeting HTT1a are developed, and that the effects of HTT-lowering strategies on the subcellular levels of all HTT transcripts and their various HTT protein isoforms are understood.

Brain microvascular endothelial cell dysfunction in an isogenic juvenile iPSC model of Huntington’s disease

Huntington’s disease (HD) is an inherited neurodegenerative disease caused by expansion of cytosine–adenine–guanine (CAG) repeats in the huntingtin gene, which leads to neuronal loss and decline in cognitive and motor function. Increasing evidence suggests that blood–brain barrier (BBB) dysfunction may contribute to progression of the disease. Studies in animal models, in vitro models, and post-mortem tissue find that disease progression is associated with increased microvascular density, altered cerebral blood flow, and loss of paracellular and transcellular barrier function. Here, we report on changes in BBB phenotype due to expansion of CAG repeats using an isogenic pair of induced pluripotent stem cells (iPSCs) differentiated into brain microvascular endothelial-like cells (iBMECs). We show that CAG expansion associated with juvenile HD alters the trajectory of iBMEC differentiation, producing cells with ~ two-fold lower percentage of adherent endothelial cells. CAG expansion is associated with diminished transendothelial electrical resistance and reduced tight junction protein expression, but no significant changes in paracellular permeability. While mutant huntingtin protein (mHTT) aggregates were not observed in HD iBMECs, widespread transcriptional dysregulation was observed in iBMECs compared to iPSCs. In addition, CAG expansion in iBMECs results in distinct responses to pathological and therapeutic perturbations including angiogenic factors, oxidative stress, and osmotic stress. In a tissue-engineered BBB model, iBMECs show subtle changes in phenotype, including differences in cell turnover and immune cell adhesion. Our results further support that CAG expansion in BMECs contributes to BBB dysfunction during HD.